S. aureus fibrinogen binding protein gene

ABSTRACT

The isolation of genes and proteins from  Staphylococcus aureus  is provided, and the nucleic acids coding for specific regions of the  S. aureus  protein are described. The nucleic acids encode proteins that can be useful as vaccines or in pharmaceutical compositions for application to prevent infection, promotion of wound healing, blocking adherence to indwelling medical devices, or diagnosis of infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.11/348,437, filed Feb. 7, 2006, now U.S. Pat. No. 7,368,112, which was acontinuation application of application Ser. No. 09/679,643, filed Oct.5, 2000, now U.S. Pat. No. 6,994,855, which was a divisional applicationof application Ser. No. 09/421,868, filed Oct. 19, 1999, now U.S. Pat.No. 6,177,084, which was a divisional application of application Ser.No. 08/293,728, filed Aug. 22, 1994, now U.S. Pat. No. 6,008,341.

FIELD OF THE INVENTION

The invention relates to the isolation of the fibrinogen binding proteingene from Staphylococcus aureus and to the use of the fibrinogen bindingprotein and antibodies generated against it for wound healing, blockingadherence to indwelling medical devices, immunization or diagnosis ofinfection.

BACKGROUND OF THE INVENTION

In hospitalized patents Staphylococcus aureus is an important cause ofinfections associated with indwelling medical devices such as cathetersand prostheses (Maki, 1982; Kristinsson, 1989) and non-device relatedinfections of surgical wounds. A recent significant increase in isolatesfrom European and US hospitals which are resistant to severalantibiotics and the potential threat of emergence of vancomycinresistance in S. aureus has reinforced the importance of developingalternative prophylactic or vaccine strategies to decrease the risk ofnosocomial infections due to S. aureus.

Initial localized infections can lead to more serious invasiveinfections such as septicemia and endocarditis. In infections associatedwith medical devices, plastic and metal surfaces become coated with hostplasma and matrix proteins such as fibrinogen and fibronectin shortlyafter implantation (Baier, 1977; Kochwa et al, 1977; Cottonaro et al,1981). The ability of S. aureus to adhere to these proteins is believedto be a crucial determinant for initiating infection (Vaudaux et al,1989, 1993). Vascular grafts, intravenous catheters, artificial heartvalves and cardiac assist devices are thrombogenic and are prone tobacterial colonization. S. aureus is the most damaging pathogen of suchinfections.

Fibrin is the major component of blood clots and fibrinogen/fibrin isone of the major plasma proteins deposited on implanted biomaterial.There is considerable evidence that bacterial adherence tofibrinogen/fibrin is of importance in initiation of device relatedinfection. (i) S. aureus adheres to plastic coverslips coated in vitrowith fibrinogen in a dose-dependent manner (Vaudaux et al, 1989) and tocatheters coated in vitro with fibrinogen (Cheung and Fischetti, 1990).(ii) The organism binds avidly via a fibrinogen bridge to plateletsadhering to surfaces in a model that mimics a blood clot or damage to aheart valve (Herrmann et al., 1993). (iii) S. aureus can adhere tocultured endothelial cells via fibrinogen deposited from plasma actingas a bridge (Cheung et al., 1991). This suggests that fibrinogen couldhave a direct role in promoting invasive endocarditis. (iv) Mutantsdefective in a global regulatory gene sar have reduced adherence tofibrinogen and have reduced infectivity in a rat endocarditis infectionmodel (Cheung et al., 1994). While this is indicative of a role foradherence to fibrinogen in initiating endocarditis it is by no meansconclusive because sar mutants are pleiotropic and could also lack otherrelevant factors.

A receptor for fibrinogen often called the “clumping factor” is locatedon the surface of S. aureus cells (Hawiger et al., 1978, 1982). Theinteraction between bacteria and fibrinogen in solution results ininstantaneous clumping of bacterial cells. The binding site for clumpingfactor of fibrinogen is located in the C-terminus of the gamma chain ofthe dimeric glycoprotein. The affinity for the fibrinogen receptor isvery high (Kd 9.6×10⁻⁹ M) and clumping occurs in low concentrations offibrinogen. It is assumed that clumping factor also promotes bacterialadhesion to solid-phase fibrinogen and to fibrin.

Clumping factor has eluded previous attempts at molecularcharacterisation. Reports of attempts to purify clumping factordescribed molecules with molecular masses ranging from 14.3 kDa to 420kDa (Duthie, 1954; Switalski, 1976; Davison and Sanford, 1982; Espersenet al., 1985; Usui, 1986, Chhatwal et al., 1987; Lantz et al., 1990) butnone were followed up with more detailed analysis. Fibrinogen is oftenheavily contaminated with IgG and fibronectin and unless specific stepswere taken to eliminate them these studies must be suspect.

More recently it has been shown that S. aureus releases several proteinsthat can bind to fibrinogen (Boden and Flock, 1989, 1992, 1994; HomonyloMcGavin et al., 1993). One of these is probably the same as the broadspectrum ligand binding protein identified by Homonylo McGavin et al.,(1993). Another is coagulase (Boden and Flock, 1989), a predominatelyextracellular protein that activates the plasma clotting activity ofprothrombin. Coagulase binds prothrombin at its N-terminus and alsointeracts with fibrinogen at its C-terminus (McDevitt et al., 1992).However, a hypothesis that the cell-bound form of coagulase is theclumping factor was disproved when coagulase-defective mutants wereshown to retain clumping factor activity (McDevitt et al., 1992). Thereis no evidence that the fibrinogen binding region of any of theseproteins is exposed on the bacterial cell surface and consequently thereis no evidence that any is clumping factor.

OBJECT OF THE INVENTION

An object of the present invention is to obtain a minimal fibrinogenbinding protein. A further objective is to obtain said protein by meansof a genetic engineering technique by using e.g. a plasmid comprising anucleotide sequence coding for said protein. A further objective is toobtain said protein by chemical synthesis. An additional objective is togenerate antisera against said protein.

SUMMARY OF THE INVENTION

The present invention relates to an isolated fibrinogen binding proteingene from S. aureus, particularly the DNA molecule having the sequenceshown in FIG. 2 and sequence ID No. 1, or a substantially similarsequence also encoding S. aureus fibrinogen binding protein.

The invention also relates to hybrid DNA molecules, e.g. plasmidscomprising a nucleotide sequence coding for said protein. Further theinvention relates to transformed host micro-organisms comprising saidmolecules and their use in producing said protein. The invention alsoprovides antisera raised against the above fibrinogen binding proteinand vaccines or other pharmaceutical compositions comprising the S.aureus fibrinogen binding protein. Furthermore the invention providesdiagnostic kits comprising a DNA molecule as defined above, the S.aureus fibrinogen binding protein and antisera raised against it.

By “substantially similar” is meant a DNA sequence which by virtue ofthe degeneracy of the genetic code is not identical with that shown inFIG. 2 and sequence ID No. 1 but still encodes the same amino-acidsequence; or a DNA sequence which encodes a different amino-acidsequence which retains fibrinogen binding protein activity eitherbecause one amino-acid is replaced with another similar amino-acid orbecause the change (whether it be substitution, deletion or insertion)does not affect the active site of the protein.

DRAWINGS

The invention will be described further with reference to the drawingsin which there is shown:

FIG. 1. Adherence of S. aureus Newman strains to fibrinogen-coated PMMAcoverslips. The number of adherent bacteria is shown as a function offibrinogen adsorbed on the coverslip. The symbol for Newman wild type isIIIIXIII. Symbols for Newman mutant strains are as follows: mutant 1,-.□.-; mutant 2, -Δ-; mutant 3, -⋄.-; mutant 4, -..-. Symbols for Newmanmutants carrying pCF16 are as follows: mutant 1, -.▪.-; mutant 2, -▴-;mutant 3, -♦-; mutant 4, -▾-.: The number of bacterial cells bound isshown as CFU (mean+/−range, n=2). In points where range bars are notvisible, the bars are smaller than the symbols.

FIG. 2 (A) Nucleotide and deduced amino acid sequence of the clfA geneof Staphylococcus aureus strain Newman. The sequence has been lodged inthe EMBL Data Library under the accession number Z18852 SAUCF. Putative−35, −10, ribosome binding site (RBS) and transcriptional stop regionsare indicated on the nucleotide sequence. For the ClfA protein, thestart of the signal peptide (S), non repeat region (A), repeat region(R), wall-spanning region (W) and membrane spanning region (M) areindicated by horizontal arrows. The LPXTG motif is underlined. (B)Schematic diagram showing the domain organization of the ClfA protein.S, signal peptide; A, non-repeat region; R, repeat region; W, wallregion; M, membrane spanning region and +, positively charged residues.The position of the LPXTG motif is indicated.

FIG. 3. Proteins purified from E. coli TBI expressing pCF17. A DNAfragment corresponding to the N-terminal half of ClfA (residues 23-550;Region A) was generated by PCR and cloned in-frame into the expressionvector pKK233-2 to generate pCF17. The N-terminal sequence was deducedfor the three fibrinogen binding proteins (105 kDa, 55 kDa and 42 kDa)purified from an induced culture of E. coli carrying pCF17 (Table 1) andthe location of each with respect to the A domain and amino acidsrepresented are indicated. Recombinant proteins which possess fibrinogenbinding activity are denoted by ++.

FIG. 4. Inhibition of adherence of strain Newman .Δ. spa tofibrinogen-coated PMMA coverslips by anti-ClfA sera and preimmune sera.The symbol for anti Region A serum N2 is -▪.- and the symbol forpreimmune serum N2 is -..-. The symbol for anti Region RWM serum C2 is-.•.-. The percentage inhibition is shown as mean+/−range, n=2. Inpoints where range bars are not visible, the bars are smaller than thesymbols.

FIG. 5. Localization of the fibrinogen binding domain of ClfA. DNAfragments corresponding to the different segments of clfA were generatedby PCR and cloned in-frame into the fusion protein expression vectorpGEX-KG. ClfA truncates were expressed as fusion proteins withglutathione S-transferase. The location of the clfA gene fragments, theamino acids represented and the length of the protein amplified are alsoindicated. The properties of the recombinant proteins are indicated.Proteins were assessed for (a) ability to bind to fibrinogen in theaffinity blotting assay (binds fg), (b) the ability of lysates toinhibit the clumping of bacteria in soluble fibrinogen (inhibitsclumping), (c) the ability of lysates to inhibit the adherence ofbacteria to solid-phase fibrinogen (inhibit adherence), and (d) theability of lysates to block neutralising antibodies (blocks Abs). ++,positive reaction; −, negative; ND, not done.

FIG. 6A. (A) Inhibition of adherence of S. aureus Newman tofibrinogen-coated coverslips by lysates containing ClfA truncates.Symbols are E. coli pCF24 uninduced lysate -.Δ-, E. coli pCF24 inducedlysate -.▴.- E. coli pCF25 uninduced lysate -.□.-, E. coli pCF25 inducedlysate -.▪.-. The percentage inhibition is shown as mean+/−range, n=2.In points where range bars are not visible, the bars are smaller thanthe symbols.

FIG. 6B. Inhibition of adherence of S. aureus Newman tofibrinogen-coated coverslips by lysates containing ClfA truncates.Symbols are E. coli pCF27 lysate -.▪.-, E. coli pCF28 lysate-.•.-, E.coli pCF29 lysate-.▴.- E. coli pCF30 lysate -.▾.-, E. coli pCF31 lysate-.♦.-. The percentage inhibition is shown as mean+/−range, n=2. Inpoints where range bars are not visible, the bars are smaller than thesymbols.

FIG. 7. Adherence of S. aureus Newman strains to PMMA coverslips coatedin vitro with fibrinogen. The number of adherent bacteria is shown as afunction of fibrinogen adsorbed on the coverslip. The symbols are,Newman wild type, -.∘.-; Newman clfA::Tn917, -.•.-. The number ofbacterial cells bound is shown as c.f.u. (mean+/−range, n=2). In pointswhere range bars are not visible, the bars are smaller than the symbols.

FIGS. 8A-8B . Adherence of S. aureus Newman strains onto segments of exvivo polymer tubing exposed to canine blood. Adherence was tested toboth ex vivo polyvinylchloride (PVC) and to ex vivo polyethylene (PE).The symbols are, Newman wild type, -.∘.-; Newman clfA::Tn917, -.•.-. Thenumber of bacterial cells bound is shown as c.f.u. (mean+/−range, n=2).In points where range bars are not visible, the bars are smaller thanthe symbols.

FIGS. 9A-9B. Adherence of S. aureus 8325-4 strains onto segments of exvivo polymer tubing exposed to canine blood. Adherence was tested toboth ex vivo polyvinylchloride (PVC) and to ex vivo polyethylene (PE).The symbols are: 8325-4 wild type, -.□.-; 83254 clfA::Tn917, -.▪-,8325-4 clfA::Tn917 (pCF4), - □-. The number of bacterial cells bound isshown as c.f.u. (mean=/−range, n=2). In points where range bars are notvisible, the bars are smaller than the symbols.

CLONING AND SEQUENCING THE CLUMPING FACTOR GENE

In view of the difficulties mentioned above with (i) obtaining purefibrinogen, (ii) the discrepancies in reported molecular weight of“clumping factor” and (iii) the diversity of different fibrinogenbinding proteins, a different approach was taken to identify theclumping factor gene involving isolating insertion mutants thatinactivated the clumping phenotype. This has been described in detail byMcDevitt et al., (1994).

Transposon Tn917 (Tomich et al., 1980) was used to generate insertionmutants that eliminated the fibrinogen clumping phenotype of S. aureusstrain Newman. The temperature sensitive plasmid pTV1ts which carriesTn917 (Youngman, 1985) was transferred into strain Newman and severaltransposon insertion banks were isolated by growing cultures at 430 inbroth containing erythromycin (to select for Tn917 after plasmidelimination). Cultures of the banks were mixed with fibrinogen, theagglutinated cells were removed and the surviving cells in thesupernatants were screened for clumping factor-deficient mutants. Fourmutants were isolated from separate banks. The Tn917 elements weretransduced into a wild-type Newman host with phage 85. In each case allthe transductants screened had inherited the clumping factor deficiencyproving that the Tn917 insertions caused the mutant phenotypes. Theclumping factor mutants expressed the same level of coagulase as thewild-type strain, further supporting the conclusion that clumping factorand coagulase are distinct entities.

The mutants were analyzed by Southern hybridization using an internalfragment of Tn917 as a probe in order to identify HindIII junctionfragments comprising transposon and flanking chromosomal sequences. Ajunction fragment from one mutant was cloned using standard techniquesin plasmid vector pUC18 (Yanisch Perron et al., 1985). A fragmentcomprising only chromosomal DNA flanking the transposon was isolatedfrom this plasmid and used in turn as a probe in a Southern blot ofgenomic DNA of Newman wild-type and each of the mutants. A HindIIIfragment of 7 kb that hybridized in Newman wild-type was missing in eachof the mutants. Genomic DNA of Newman wild-type was cleaved with HindIIIand ligated with plasmid pUC18 cut with the same enzyme and transformedinto E. coli TBI (Yanisch-Perron et al., 1985). Transformants werescreened by colony hybridisation using the junction fragment probe.Plasmid pCF3 (pUC18 carrying the 7 kb HindIII fragment) was isolated.Plasmid pCF3 was deposited at the NCIMB Aberdeen, Scotland on Jul. 2,1998 under the Accession No. NCIMB40959, such deposit complying with theterms of the Budapest Treaty.

The 7 kb HindIII fragment was subcloned into pCL84, a single copynon-replicating vector which integrates into the chromosome of S. aureus(Lee et al., 1991), forming pCF16. pCF16 was transformed into S. aureusstrain CYL316 (Lee et al., 1991) selecting for tetracycline resistance.The integrated plasmid was then transduced with phage 85 into each ofthe Newman clf mutants. In a microtitre clumping assay the Newmanmutants were completely devoid of activity even at the highestconcentrations of fibrinogen, whereas the wild-type had a titre of 2048and could interact productively with very low concentrations offibrinogen. The integrated single copy plasmid pCF16 restored theclumping activity of each of the mutants to the same level as that ofthe parental strain. Thus the HindIII fragment must express a functionalprotein which complements the clumping deficiency of the mutants.

S. aureus Newman adhered to solid-phase fibrinogen coated ontopolymethylmethacrylate (PMMA) coverslips in a concentration dependentmanner (FIG. 1). Each clf mutant showed drastic reduction in adherence.This was restored to the level of the parental strain by pCF16. Thisdata shows that the ability of Newman to form clumps in solublefibrinogen correlates with bacterial adherence to solid-phasefibrinogen.

Fragments from the 7 kb HindIII fragment in pCF3 were subcloned intopGEM7 Zf(+) (Promega). The smallest fragment which still expressed thefibrinogen binding activity was a 3.5 kb HindIII-KpnI fragment which iscontained in plasmid pCF10 which was deposited at the NationalCollections of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland,in September, 1994, and which was accorded Accession No. 40674. The DNAsequence of this fragment was obtained using standard techniques and hasbeen lodged in the EMBL Data Library under the accession number Z18852SAUCF. A single open reading frame of 2799 bp was identified (FIG. 2Aand Sequence ID No. 1). The orf is called clfA and the gene product theClfA protein. The predicted protein is composed of 933 amino acids(molecular weight 97,058 Da, see Sequence ID Nos. 1 and 2). A putativesignal sequence of 39 residues was predicted. The predicted molecularweight of the mature protein is 92 kDa. Following the signal sequence isa region of 520 residues (Region A) which precedes a 308 residue region(region R) comprising 154 repeats of the dipeptide serine-aspartate(FIGS. 2A and 2B and Sequence ID No. 2). The C terminus of ClfA hasfeatures present in surface proteins of other Gram positive bacteria(Schneewind et al., 1993) that are responsible for anchoring the proteinto the cell wall and membrane: (i) residues at the extreme C-terminusthat are predominantly positively charged, (ii) a hydrophobic regionwhich probably spans the cytoplasmic membrane and (iii) the sequenceLPDTG which is homologous to the consensus sequence LPXTG that occurs inall wall-associated proteins of Gram positive bacteria. This stronglysuggests that ClfA is a wall-associated protein and that the N terminalpart is exposed on the cell surface.

It is not obvious from the primary structure of ClfA or by comparison ofClfA with other ligand binding proteins of S. aureus (fibronectinbinding protein, Signas et al., 1989; collagen binding protein, Patti etal., 1992) which part of ClfA interacts with fibrinogen.

Results

(1) Purifying the N-Terminal Half of the Fibrinogen Receptor (ClfA)

A DNA fragment corresponding to the N-terminal half of ClfA (residues23-550; Region A) was generated by polymerase chain reaction (PCR) andcloned in-frame into the expression vector pKK233-2 (Amann and Brosius,1985) to generate pCF17 (FIG. 3). Expression of recombinant Region A wasinduced by adding isopropyl Beta-D-thiogalactoside (IPTG) to exponentialcultures. Induced cultures contained two proteins of 105 kDa and 55 kDawhich reacted with fibrinogen in a Western ligand blotting assay. Afibrinogen-Sepharose 4B column was made by the method recommended by themanufacturer (Pharmacia). A sample of an induced culture containingthese fibrinogen binding proteins was passed into the fibrinogenSepharose 4B column. Four proteins were eluted: −105 kDa, 55 kDa, 42 kDaand 75 kDa (trace amounts). In a separate purification experiment, the42 kDa protein was purified to homogeneity. Only the 105 kDa, 55 kDa and42 kDa proteins bound to fibrinogen in the Western ligand blottingassay. The N-terminal sequence of these proteins was determined (Table1). The 75 kDa protein was present in trace amounts (1-2 pmoles) and isnot related to ClfA. The three predominant proteins bound to fibrinogenin the Western blotting assay and are related to the region A (see FIG.3). The 105 kDa protein represents the intact Region A while the 55 kDaand 42 kDa proteins are breakdown products. The apparent molecularweights of the native region A and breakdown products of region A aremuch higher than that predicted from the DNA sequence (Table 1).

(2) Antibodies to the Region A of the ClfA Protein (Residues 23-550)

A rabbit was immunized with 30 micro g of a mixture of the 105 kDa, 75kDa, 55 kDa and 42 kDa proteins along with Freund's complete adjuvant.The immune sera was called N2. One rabbit was also immunized with 18micro g of the purified 42 kDa ClfA truncate and the immune serum forthis was called N3. Bacterial interaction with fibrinogen can bemeasured by a quantitative clumping titration assay (Switalski, 1976).In this assay, doubling dilutions of a fibrinogen solution (1 mg/ml) aremixed in a microtitre dish with a suspension of 2×10⁷ cells for 5 minwith gentle shaking. A standard clumping concentration of fibrinogen wasdefined as 2 times. the titre. To this was added varying amounts of theanti-ClfA serum to measure the minimum inhibitory concentration thatstops the clumping reaction (Table 2). Both N2 and N3 sera were potentinhibitors of the clumping of bacteria. Preimmune sera did not inhibitthe clumping of bacteria. N2 sera also had a potent inhibitory activityon bacterial adhesion to surface-bound fibrinogen in the coverslip assay(McDevitt et al., 1992, 1994), expressing 95% inhibition at 1 micro gprotein/ml (FIG. 4). Preimmune sera did not have any inhibitory activityeven at a protein concentration of 100 micro g/ml (FIG. 4). In addition,antisera raised against regions R, W and M (C2) (see section 4 below)failed to inhibit adherence even at 100 micro g/ml (FIG. 4).

(3) Localization of the Fibrinogen Binding Domain of the ClfA Protein

DNA fragments corresponding to the Region A of ClfA (residues 23-550)and C terminal regions R, W and M (residues 546-933) were generated byPCR (standard conditions,) and cloned in-frame into the fusion proteinexpression vector PGEX-KG (Guan and Dixon, 1991) to generate pCF24 andpCF25 respectively (see FIG. 5). These ClfA truncates were expressed asfusion proteins with glutathione S-transferase. An induced lysate of E.coli pCF24 (residues 23-550) expressed a fusion protein that bound tofibrinogen in a Western affinity blotting assay with peroxidase labelledfibrinogen (FIG. 5). In addition, this lysate inhibited the clumping ofbacteria with soluble fibrinogen in the clumping assay (Table 3 and FIG.5) and also inhibited the adherence of bacteria to immobilizedfibrinogen in the coverslip assay in a dose dependent fashion (FIG. 6A).A lysate of E. coli pCF25 (residues 546-933) induced with IPTG expresseda fusion protein that failed to bind to fibrinogen in the Westernblotting assay (FIG. 5). In addition, this lysate did not inhibit theclumping of bacteria in the clumping assay (Table 3) and did not inhibitadherence to immobilized fibrinogen in the adherence assay (FIG. 6A).Uninduced lysates from both pCF24 and pCF25 failed to inhibit bothclumping and adherence (Table 3 and FIG. 6A).

A synthetic peptide (SDSDSDSDSDSDGGGC, Sequence ID No. 16) designed tomimic the C-terminal repeat region of ClfA failed to inhibit theclumping of bacteria in the clumping assay when up to 107 micro g/ml wastested. In addition, the synthetic peptide failed to inhibit theadherence of bacteria in the adherence assay even at a concentration of100 micro g/ml. Taken together, this data suggests that the fibrinogenbinding domain of ClfA is located in the A domain rather than in theregions R, M, and W. It confirms the data in Table 1 which dealt withpurifying fibrinogen binding proteins expressed from pCF17 and alsosuggests that an N-terminal ClfA protein can act both as a potentinhibitor of cell clumping in fibrinogen and also as a potent inhibitorof the adherence of bacteria to fibrinogen coated surfaces.

The fibrinogen binding domain was further localized within region A.Segments of region A were amplified by PCR and cloned into the PGEX-KGvector. Lysates from IPTG-induced cultures were examined for thepresence of fibrinogen binding fusion proteins, for the ability toinhibit the clumping of bacteria in the fibrinogen clumping assay andfor the ability to inhibit adherence to immobilized fibrinogen in theadherence assay. The fusion protein of pCF31 (residues 221-550) was thesmallest truncate that still expressed a fibrinogen binding activity(FIG. 5). It is almost identical in composition to the purified 42 kDaprotein (residues 219-550) described above. The fusion proteins frompCF27, pCF28, pCF29 and pCF30 all failed to bind to fibrinogen in theWestern affinity blotting assay, despite reacting with antibodiesgenerated against the A domain of ClfA (FIG. 5). In addition, a lysatecontaining the fusion protein expressed by pCF31 was the only one toinhibit the fibrinogen clumping reaction (Table 3) and to inhibit theadherence of bacteria to immobilized fibrinogen in the adherence assay(FIG. 6B). These results suggest that the fibrinogen binding site isquite extensive or that its correct conformation is determined byflanking sequences.

An antibody neutralization assay was adopted to help localize furtherthe active site within residues 221-550. This assay was conducted with aprotein A negative deletion mutant of S. aureus strain Newman (Patel etal., 1987) to prevent non specific reaction with IgG. Polyclonalantibodies raised against the A region of ClfA (N2) inhibited theclumping of bacteria in soluble fibrinogen (see section 2 above). In thestandard clumping assay with the clumping concentration at 2 times. thetitre, the concentration of lysates that blocked the inhibitory activityof 4.68 micro g of serum (2 times the inhibitory concentration, Table 2)was determined. The lysates containing ClfA fusion proteins were assayedfor their ability to neutralize the inhibiting activity of theantibodies. Truncates containing the active site might be able to adsorbout antibodies generated against the active site and thus neutralize theblocking effect on cell clumping. The lysates containing proteinsexpressed by pCF24 and pCF31 neutralized the inhibiting activity of theantibodies while a lysate containing the fusion protein expressed bypCF25 (Region R, W and M) did not inhibit (Table 4). Lysates containingsmall fusion proteins expressed by pCF30 were able to neutralize theinhibiting activity of antibodies while lysates containing fusionproteins expressed by pCF27 and pCF29 had no such activity (Table 4).Taken together this suggested that the active site is located in a 218residue region between residues 332 and 550.

(4) Antibodies to the C-Terminal Half of the ClfA Protein (Residues546-933)

The fusion protein present in a lysate of E. coli pCF25 (residues546-933) induced with IPTG was purified to homogeneity by usingglutathione sepharose-affinity chromatography as described by Guan andDixon, (1991). A rabbit was immunized with 20 micro g of the fusionprotein along with Freund's complete adjuvant. The immune sera wascalled C2. This serum failed to inhibit the clumping of bacteria in theclumping assay (Table 2) and also failed to inhibit bacterial adhesionto surface bound fibrinogen in the coverslip assay even at 100 microg/ml (FIG. 4).

(5) Identification of the Native Fibrinogen Receptor

Proteins released from the cell wall of S. aureus strains and a lysateof E. coli expressing the cloned clfA gene were studied by Westernimmunoblotting with anti ClfA antibodies in order to identify ClfAprotein(s). A lysate of E. coli TB1 (pCF3) (carrying the cloned clfAgene) contained several immunoreactive proteins . The largest of thesewas ca. 190 kDa. The smaller protein are probably derivatives caused byproteolysis. S. aureus strain Newman also expresses a ca. 190 kDaimmunoreactive protein . A smaller immunoreactive protein of ca. 130 kDawas also detected and is probably also caused by proteolysis. Despitethe presence of protease inhibitors and studying proteins released fromcells harvested at different stages in the growth cycle (frommid-exponential to late stationary), two proteins of these sizes werealways present (data not shown). Both proteins were absent in extractsof the clumping factor negative transposon insertion mutant of Newmanindicating that they are products of the clfA gene.

Previously we reported the size of the ClfA protein to be ca. 130 kDa(McDevitt et al., 1994) in an affinity blotting assay with fibrinogenand peroxidase labelled anti-fibrinogen antibodies. Our currentimmunoblotting assay is much more sensitive than the affinity blottingassay. In addition, we now know that the ClfA protein is very sensitiveto degradation. Indeed the predominant immunoreactive protein detectedin samples from both E. coli TB1 (pCF3) and S. aureus strain Newmanwhich have been frozen and thawed more than twice is 130 kDa indicatingthat the ca. 190 kDa protein is labile (data not shown). Thus, the ca.130 kDa protein detected in the affinity blotting assay is most probablya smaller derivative of ClfA. The apparent size of the native ClfAprotein of strain Newman appears to be ca. 190 kDa. This is double thatpredicted from the DNA sequence, but this might be due to the unusualstructure and is consistent with the aberrantly high apparent molecularweight of recombinant proteins (Table 1). The recombinant N-terminalRegion A protein expressed by E. coli pCF17 also had an unexpectedlyhigh apparent molecular weight.

(6) Surface Localization of the ClfA Protein by ImmunofluorescentMicroscopy

Anti-ClfA region A sera (N2) was used to confirm that Region A of ClfAis exposed on the bacterial cell surface. Protein A-deficient mutants ofNewman and Newman clfA::Tn917 (clumping factor transposon insertionmutant) were isolated by transducing the .Δ spa::Tc^(r) mutation from8325-4 .Δ .spa::Tc^(r) to strains Newman and Newman clfA::Tn917 usingphage 85. Protein A-deficient mutants were used to prevent non-specificinteraction with rabbit IgG. Cells from overnight cultures of strainsNewman .Δ spa::Tc^(r) and Newman .Δ spa::Tc^(r) clfA::Tn917 were dilutedto As60=0.6-1.0 and fixed to glass slides using gluteraldehyde. Theslides were then incubated in anti-ClfA region A serum (N2, 1 in 200)followed by fluorescein conjugated swine anti-rabbit serum (Dakopatts, 1in 40). The cells were studied for fluorescence by microscopy (Nowickiet al., 1984). Newman.sub..Δ spa: :Tc^(r) cells fluoresced whileNewman.Δ spa::Tc^(r) clfA::Tn917 cells did not. This confirmed thatregion A of ClfA is exposed on the cell surface of wild-type Newman andthat this ClfA protein is absent in the clumping factor deficientmutant.

(7) Role of the Fibrinogen Receptor in Adherence to In Vitro- and ExVivo-Coated Polymeric Biomaterials

A mutant of strain Newman defective in the clumping factor(clfAl::Tn917) and a complemented mutant bearing pCF16 were studied foradherence properties to biomaterials coated in vitro with fibrinogen andto ex vivo biomaterial. A canine arteriovenous shunt has been developedas a model to study plasma protein adsorption onto intravenous cathetersfrom short-term blood-biomaterial exposures and to identify hostproteins promoting adhesion of Staphylococcus aureus (Vaudaux et al.,1991).

S. aureus strain Newman adheres strongly (in a concentration dependentfashion) to polymethylmethacrylate (PMMA) coverslips coated in vitrowith canine fibrinogen (FIG. 7). In contrast, the fibrinogen receptormutant was significantly defective (>95%) in its ability to adhere tothe canine fibrinogen coated coverslips (FIG. 7). In the ex vivo model,either polyethylene or polyvinyl chloride tubing was exposed to canineblood for 5, 15 or 60 min at a flow rate of 300 ml/min, then flushed inphosphate buffered saline (PBS), cut into 1.5 cm segments andpreincubated in 0.5% albumin in PBS to prevent non-specificstaphylococcal attachment. Then, each segment was incubated with 4×10⁶CFU/ml of [3H]thymidine-labelled S. aureus for 60 min at 37° C. in an invitro adherence assay. When compared with the wild-type strain Newman,the fibrinogen receptor mutant strain showed a strong decrease (>80%) inattachment to ex vivo polyvinyl chloride and polyethylene tubings (FIG.8A-B). In addition, strain 8325-4 (which binds poorly tofibrinogen-coated coverslips in vitro and to the ex vivo polymertubings) showed a significant increase in its ability to adhere to thetwo different ex vivo polymer tubings when complemented with a plasmid(pCF4) expressing the fibrinogen receptor gene (FIG. 9A-B).

The data shows that fibrinogen is the major plasma protein in ashort-term blood material interaction to promote staphylococcaladherence and the possession of the fibrinogen receptor is a majordeterminant in the ability of S. aureus to adhere to ex vivobiomaterials.

(8) Role of the Fibrinogen Receptor in the Pathogenesis of ExperimentalEndocarditis

S. aureus strain Newman, the fibrinogen receptor mutant strain of Newman(clfA::Tn917) and a fibrinogen receptor mutant complemented with theclfA+ integrating plasmid pCF16 were compared in a previously describedmodel of experimental endocarditis (Garrison and Freedman, 1970). Thisrat model investigates the early events in experimental endocarditiswith catheter-induced aortic vegetations (Veg). Groups of >/−8 rats werechallenged with an inoculum that resulted in 90% of vegetations beingcolonized by the wild-type organism (ID90). Animals were injectedintravenously with the same inocula of Newman clfA and Newman clfA(pCF16). Animals were killed 12 hours after inoculation and quantitativecultures of the blood, spleen and Veg were performed. Table 5 shows thepercentage of rats infected.

The data show that a mutant lacking the fibrinogen receptor wassignificantly less able to infect the catheter-induced aorticvegetations (decrease of 49%) when compared with the wild type strainNewman. In addition, the complemented strain had restored infectivity.The fact that all three strains infected the spleens with similarnumbers suggests that the presence or absence of the fibrinogen receptorinterfered specifically with bacterial colonization of thecatheter-induced aortic vegetation.

This model strongly implicates the fibrinogen receptor as an importantadhesin in the pathogenesis of S. aureus endocarditis and othercardiovascular infections associated with intravenous catheters,artificial heart valves and intravenous shunts.

Uses of the Invention

1. The fibrinogen binding protein or fragment containing the fibrinogenbinding region can be used as a vaccine to protect against human andanimal infections caused by S. aureus. For example, the fibrinogenbinding protein or fragment containing the fibrinogen binding region canbe used as a vaccine to protect ruminants against mastitis caused by S.aureus infections.

2. Polyclonal and monoclonal antibodies raised against the fibrinogenbinding protein or a fragment containing the fibrinogen binding domaincan be used to immunise passively by intravenous injection againstinfections caused by S. aureus.

3. The fibrinogen binding protein or an active fragment can beadministered locally to block S. aureus from colonising and infecting awound.

4. The antibody against the fibrinogen binding protein can beadministered locally to prevent infection of a wound.

5. The fibrinogen binding protein or an active fragment or antibodiesagainst the fibrinogen binding protein can be used to block adherence ofS. aureus to indwelling medical devices such as catheters, replacementheart valves and cardiac assist devices.

6. The fibrinogen binding protein or an active fragment or antibodiesagainst the fibrinogen binding protein can be used in combination withother blocking agents to protect against human and animal infectionscaused by S. aureus.

7. The fibrinogen binding protein can be used to diagnose bacterialinfections caused by S. aureus strains. The fibrinogen binding proteincan be immobilised to latex or Sepharose (Trade Mark), and seracontaining antibodies are allowed to react; agglutination is thenmeasured.

8. The fibrinogen binding protein can be used in an ELISA test.

9. DNA gene probe for the fibrinogen binding protein for ELISA tests.

10. Antibodies to the fibrinogen binding protein can be used to diagnosebacterial infections caused by S. aureus strains.

TABLE 1 ClfA proteins. Protein mol. wt. ClfA apparent* predicted@N-terminal sequence residues 105 kDa  57 kDa VGTLIGFGLL, SEQ ID NO: 1723-32 75 kDa ND GDIIGID, SEQ ID NO: 18 not related 55 kDa 44 kDaMNQTSNETTFNDTNTV, SEQ ID NO: 19 143-157 42 kDa 36 kDaAVAADAPAAGTDITNQLT, SEQ ID NO: 20 0 220-237 Native ClfA 190 kDa  92 kDa*determined from migration on SDS-PAGE and Western blotting. @predictedfrom the amino acid sequence ND not determined.

TABLE 2 Inhibition of clumping with anti-ClfA sera. Inhibitingconcentration* Sera (micro g) N2 2.34 N3 2.34 Preimmune N2 >300.00Preimmune N3 >300.00 C2 >300.00 *Average of 3 experiments.

TABLE 3 Inhibition of clumping with lysates containing truncated ClfAproteins. Inhibiting concentration* Lysate (micro g) pCF24 9.37pCF25 >300.00 pCF24 Uninduced >300.00 pCF25 Uninduced >300.00pCF27 >300.00 pCF28 >300.00 pCF29 >300.00 pCF30 >300.00 pCF31 9.37*Average of 3 experiments.

TABLE 4 The ability of lysates to block the inhibiting effect ofanti-ClfA N2 sera on cell clumping. Blocking concentration* Lysate(micro g) pCF24 1.17 pCF25 >75.00 pCF27 >75.00 pCF28 >75.00 pCF29 >75.00pCF30 2.34 pCF31 2.34 *Average of 3 experiments.

TABLE 5 Experimental endocarditis Newman Newman clfA::Tn917 % infectedNewman clfA::Tn917 pCF16 clfA+ vegetation 84% 43%* 83% blood cultures70% 30%* 50% spleen (x log CFU/g) 3.16 3.11 3.59 *p = 0.05 when comparedto other groups

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1. A method of immunizing a mammal comprising administering to saidmammal an immunogenic amount of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, amino acids 23 to 550 of SEQ ID NO:2,143 to 550 of SEQ ID NO:2, 219 to 550 of SEQ ID NO:2, 332 to 550 of SEQID NO:2, 420 to 550 of SEQ ID NO:2, 546 to 933 of SEQ ID NO:2, 332 to425 of SEQ ID NO: 2, 23 to 424 of SEQ ID NO:2, 23 to 308 of SEQ ID NO:2,40 to 559 of SEQ ID NO:2 and 221 to 550 of SEQ ID NO:2.
 2. A method ofraising an immune response against S. aureus in a human or animalpatient comprising administering to said patient a compositioncomprising an immunogenic amount of an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, amino acids 23 to 550 of SEQ IDNO:2, 143 to 550 of SEQ ID NO:2, 219 to 550 of SEQ ID NO:2, 332 to 550of SEQ ID NO:2, 420 to 550 of SEQ ID NO:2, 546 to 933 of SEQ ID NO:2,332 to 425 of SEQ ID NO: 2, 23 to 424 of SEQ ID NO:2, 23 to 308 of SEQID NO:2, 40 to 559 of SEQ ID NO:2 and 221 to 550 of SEQ ID NO:2, and apharmaceutically acceptable carrier or adjuvant.